Fluorescent material for near-infrared two-region imaging as well as preparation method and application of fluorescent material

Abstract

The invention provides a fluorescent material for near-infrared two-region imaging as well as a preparation method and application of the fluorescent material. The fluorescent material is nanoparticles and comprises a D-A-D type compound with a structure as shown in a formula (I). The fluorescent material containing the D-A-D type compound with the structure as shown in the formula (I) has good application in near-infrared two-region imaging. The D-A-D type compound has the maximum emission wavelength at about 1000 nm under the laser wavelength of 884 nm, and the fluorescence quantum yield is relatively high. The fluorescent material disclosed by the invention has a good imaging effect in vitro and in vivo, and shows a wide application prospect in the field of near-infrared two-region imaging. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a fluorescent material for near-infrared second-zone imaging, a preparation method thereof, and an application thereof. Background Art

[0002] Medical imaging technology enables visualization of the internal structure of organisms and can dynamically monitor their physiological activities and pathological changes. It plays an irreplaceable role in clinical diagnosis, precise navigation of surgical operations, and scientific assessment of patient prognosis. Compared with classic imaging technologies such as magnetic resonance imaging, X-ray tomography, and ultrasound, fluorescence imaging technology has advantages in low cost, high sensitivity, high resolution, and the ability to detect and locate biological molecules in real time. Among them, thanks to the reduction in light scattering and tissue autofluorescence interference caused by the increase in wavelength, near-infrared second region (1000-1700nm) fluorescence imaging shows a stronger penetration depth and higher signal-to-noise ratio, making it a highly promising technology in the field of biological imaging.

[0003] With the rapid development of near-infrared second-zone imaging technology, researchers have successively explored and designed a variety of fluorescent materials to meet the wide range of needs in the field of biological imaging. Among them, compared with inorganic nanomaterials such as semiconductor quantum dots and rare-earth-doped nanoparticles, organic fluorophores have unique advantages. They not only have flexible and adjustable fluorescence properties, but also exhibit good biosafety and biocompatibility. In order to obtain a higher fluorescence quantum yield, a fluorescent material with a DAD structure (a conjugated molecular configuration composed of alternating electron donors (Donor, D) and electron acceptors (Acceptor, A)) is synthesized by introducing groups with strong electron donor and acceptor capabilities. This material has great application potential and development prospects in the fields of vascular imaging and tumor imaging. Summary of the Invention

[0004] To address the challenges of the prior art, the present invention provides a fluorescent material for near-infrared second-zone imaging, as well as its preparation method and application. The DAD (donor-acceptor-donor) compound provided by the present invention enables high-resolution near-infrared second-zone vascular imaging at an excitation wavelength of 884 nm.

[0005] The fluorescent material for near-infrared second-zone imaging provided by the present invention is a nanoparticle comprising a DAD-type compound having a structure shown in formula (I):

[0006]

[0007] In formula (I), R1, R2, R3, and R4 are each independently selected from a hydrogen atom or a C 1-10 alkyl;

[0008] R5, R6, R7, R8 groups are independently selected from hydrogen atoms, halogen atoms, nitro groups, cyano groups, C 1-6 Halogenated alkyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylcarbonyl, C 1-6 Alkoxycarbonyl, or R6 and R7 together form a 5-14 membered aromatic ring or heteroaromatic ring fused to a benzene ring; wherein the heteroaromatic ring contains 1-3 heteroatoms independently selected from N, O or S; the aromatic ring or heteroaromatic ring is optionally substituted by 1-3 substituents, each of which is independently selected from halogen, nitro, cyano, C 1-6 Halogenated alkyl, C 1-6 Alkylsulfonyl, C 1-6 Alkoxycarbonyl;

[0009] n is a positive integer from 1 to 4.

[0010] According to an embodiment of the present invention, R1, R2, R3, and R4 are each independently selected from a hydrogen atom or a C 1-8 alkyl;

[0011] R5, R6, R7, R8 groups are independently selected from hydrogen atoms, halogen atoms, nitro groups, cyano groups, C 1-4 Halogenated alkyl, C 1-4 Alkylsulfonyl, C 1-4 Alkylcarbonyl, C 1-4 Alkoxycarbonyl, or R6 and R7 together form a 5-14 membered aromatic ring or heteroaromatic ring fused to a benzene ring; wherein the heteroaromatic ring contains 1-3 heteroatoms independently selected from N, O or S; the aromatic ring or heteroaromatic ring is optionally substituted by 1-3 substituents, each of which is independently selected from halogen, nitro, cyano, C 1-4 Halogenated alkyl, C 1-4 Alkylsulfonyl, C 1-4 Alkoxycarbonyl;

[0012] n is a positive integer from 1 to 2.

[0013] The DAD-type compound of the structure represented by the above formula (I) is prepared by a method comprising the following steps: reacting a compound represented by formula II with a compound represented by formula III under alkaline conditions to obtain a DAD-type compound represented by formula (I);

[0014]

[0015] In formula II, R1, R2, R3, R4, and n are defined as R1, R2, R3, R4, and n in formula (I);

[0016] In formula III, R5, R6, R7, and R8 are defined the same as R5, R6, R7, and R8 in formula (I);

[0017] In the above method, the reaction is carried out in an inert atmosphere, specifically a nitrogen atmosphere;

[0018] The alkaline conditions are provided by an organic base, specifically pyridine, N,N-dimethylaminopyridine, triethylamine, or N,N-diisopropylethylenediamine;

[0019] The molar ratio of the compound represented by formula II to the compound represented by formula III may be 1:(2-5), specifically 1:5;

[0020] The reaction temperature may be 85-100°C and the reaction time may be 12-24 hours;

[0021] The reaction is carried out in an organic solvent, and the organic solvent can be specifically chloroform;

[0022] The above method further comprises removing the solvent by rotary evaporation under reduced pressure after the reaction is completed, and then purifying the mixture by silica gel column chromatography to obtain a DAD-type compound with the structure represented by formula (I), wherein the eluent used for the silica gel column chromatography purification is chloroform.

[0023] In the above method, the compound represented by formula II is prepared by a method comprising the following steps: reacting the compound represented by formula IV with the compound represented by formula V in an inert atmosphere in the presence of a palladium catalyst to obtain the compound represented by formula II;

[0024]

[0025] In formula IV, R1, R2, R3, and R4 are defined as R1, R2, R3, and R4 in formula (I);

[0026] In formula V, n is defined as in formula (I).

[0027] In the above method, the reaction is carried out in an organic solvent, and the organic solvent can specifically be ultra-dry toluene;

[0028] The inert atmosphere is a nitrogen atmosphere;

[0029] The palladium catalyst may be selected from at least one of palladium acetate, palladium dichloride, tetrakistriphenylphosphine palladium, bis(triphenylphosphine)palladium dichloride, Pd2(dba)3 / PPh3, and Pd(OAc)2 / PPh3;

[0030] The molar ratio of the compound represented by formula IV, the compound represented by formula V, and the palladium catalyst can be 1:(2-5):(0.1-5), specifically 1:2.5:0.1;

[0031] The reaction temperature may be 80-120°C, specifically 110°C, and the reaction time may be 12-24 hours;

[0032] The above method further comprises the steps of removing the solvent by rotary evaporation under reduced pressure after the reaction is completed and purifying the compound by silica gel column chromatography to obtain the compound represented by formula II.

[0033] The silica gel column chromatography was used as the eluent for purification: petroleum ether / dichloromethane (volume ratio of 2:1).

[0034] The fluorescent material for near-infrared second-zone imaging further comprises liposomes, and the liposomes are used to encapsulate the DAD-type compound having the structure shown in formula (I);

[0035] The mass ratio of the DAD-type compound having the structure shown in formula (I) to the liposome can be 1:5-1:10.

[0036] The liposome can specifically be distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and its molecular weight is 2000-5000.

[0037] The use of the DAD-type compound having the structure shown in the above formula (I) and the fluorescent material containing the DAD-type compound having the structure shown in the above formula (I) as and / or in the preparation of products for near-infrared second-zone imaging also falls within the scope of protection of the present invention.

[0038] In the application, the product can be used for in vivo near-infrared zone II vascular imaging.

[0039] The beneficial effects of the present invention are as follows:

[0040] The fluorescent material provided by the present invention, comprising a DAD-type compound having the structure represented by formula (I), has promising applications in near-infrared (NIR) II imaging. The DAD (donor-acceptor-donor) compound provided by the present invention exhibits a maximum emission wavelength of approximately 1000 nm at a laser wavelength of 884 nm and exhibits a high fluorescence quantum yield. The fluorescent material described in the present invention exhibits excellent imaging effects both in vitro and in vivo. In applications involving vascular imaging, it can be directly used for high-resolution vascular imaging in living mice via tail vein injection, demonstrating broad application prospects in the field of NIR II imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The synthetic route of the DAD-type compound of the structure shown in formula (I) of the present invention is shown in FIG.

[0042] Figure 2 These are the hydrogen and carbon nuclear magnetic resonance spectra of compound 3a and PDT-EDOT-2F molecules prepared in Example 1 of the present invention in deuterated chloroform.

[0043] Figure 3 TEM characterization of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention.

[0044] Figure 4 The hydrodynamic radius and surface charge of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention are characterized.

[0045] Figure 5 These are the near-infrared fluorescence absorption and emission spectra of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention.

[0046] Figure 6 This is the fluorescence quantum yield characterization of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention.

[0047] Figure 7 This is the characterization of the near-infrared fluorescence effect of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention in water, PBS, and FBS solutions.

[0048] Figure 8 This is a near-infrared zone II angiography image of the PDT-EDOT-2F nanoparticles prepared in Example 1 of the present invention in mice. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0050] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0051] Example 1. Preparation of PDT-EDOT-2F Nanoparticles

[0052]

[0053] Among them, EH is

[0054] PDT-EDOT-2F was prepared according to the following flow chart;

[0055]

[0056] a) Compounds 1a and 2a were synthesized according to literature reports (synthesis reference of 1a: Science Advances, 10(23), eadm9631; 2024; synthesis reference of 2a: The Journal of Organic Chemistry, 71(18), 6734–6741; 2006).

[0057] b) Synthesis of compound 3a: Compound 1a (40.0 mg, 0.04 mmol, 1 equivalent) and 2a (25.1 mg, 0.1 mmol, 2.5 equivalents) were placed in a 15 ml pressure tube, nitrogen was replaced three times, and then 10 ml of ultra-dry toluene was added. Nitrogen was bubbled into the solution with stirring for 10 minutes, and tetrakistriphenylphosphine palladium (4.7 mg, 0.004 mmol, 0.1 equivalent) was added under a nitrogen atmosphere. After sealing the tube, the pressure tube was placed in a 110-degree oil bath and stirred overnight. After the reaction solution was cooled to room temperature, the solvent was removed by vacuum rotary evaporation, and the target product 3a (30.3 mg, 75%) was purified by silica gel column chromatography using petroleum ether / dichloromethane (volume ratio of 2 / 1) as eluent. The nuclear magnetic resonance hydrogen spectrum (300 MHz) of compound 3a in deuterated chloroform is as shown below. Figure 2 shown.

[0058] NMR analysis results are as follows: 1H NMR (300 MHz, CDCl3): δ (ppm) 9.87 (s, 2H), 7.34 (s, 2H), 4.44 (d, J = 1.47 Hz, 8H), 1.88-1.83 (m, 8H), 1.10-0.60 (m, 60H). 13C NMR (101 MHz, CDCl3): δ (ppm) 178.81, 158.28, 155.30, 148.73, 142.08, 135.96, 132.80, 125. 52,121.01,113.69,65.34,64.79,53.38,53.34,42.09,41.96,41.86,35.29,35.15,34.33,34.01,28.47,28.16,28.05,27.55,27.11,27.02,23.13,23.11,22.95,14.07,14.04,14.02,10.61,10.56,10.15,10.03.

[0059] Mass spectrometry analysis results: HRMS (MALDI-TOF): calcd. for C58H80O6S4[M]+1000.4832, found 1000.4825.

[0060] c) Synthesis of compound 4a (PDT-EDOT-2F): Compound 3a (30 mg, 0.03 mmol, 1 equivalent) was placed in a 15 ml pressure tube, nitrogen was replaced three times, and then 10 ml of chloroform was added. Nitrogen was blown for 10 minutes, and then difluoroindigo ketone (34.5 mg, 0.15 mmol, 5 equivalents) was added. 0.1 ml of pyridine was added, and the tube was sealed and placed in an 85 ° C oil bath and stirred overnight. After the reaction solution was cooled to room temperature, the solvent was removed by vacuum rotary evaporation, and the target product 4a (30.3 mg, 75%) was purified by silica gel column chromatography using chloroform as the eluent. The nuclear magnetic resonance hydrogen spectrum (400 MHz) of compound 4a in deuterated chloroform is as follows Figure 2 shown.

[0061] The NMR analysis results of compound 4a (PDT-EDOT-2F) are as follows: 1H NMR (400 MHz, CDCl3): δ (ppm) 8.97 (s, 2H), 8.50-8.46 (m, 2H), 7.67 (s, 2H), 7.63-7.26 (m, 2H), 4.58-4.57 (d, J = 4.45 Hz, 4H), 4.51-4.50 (d, J = 4.58 Hz, 4H,), 1.94 (m, 8H), 1.25-0.64 (m, 60H). 13C NMR (176MHz, CDCl3): δ (ppm) 186.57, 160.86, 160.84, 158.48, 154.71, 153.22, 152.35, 146.10, 136.69,134.67,134.18,130.57,123.22,117.44,115.24,115.01,114.77,114.65,113.73,112 .08,111.96,66.41,66.16,64.78,53.68,53.44,42.24,35.41,29.71,29.22,28.46,28.42,28.13,28.03,28.01,27.50,27.47,27.22,27.13,23.13,22.98,14.10,14.04,10.54,10.19,10.09.

[0062] Mass spectrometry analysis results: HRMS (MALDI-TOF): calcd. for C82H84F4N4O6S4[M]+1424.5204; found: 1424.5192.

[0063] d) Liposome coating: 0.4 mg of PDT-EDOT-2F and 4 mg of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG, molecular weight 2000) were dissolved in 2 mL of tetrahydrofuran, sonicated, and then 9 mL of ultrapure water was added. The mixture was stirred at room temperature for 48 h to obtain the fluorescent material (PDT-EDOT-2F nanoparticles).

[0064] Example 2: Performance Characterization and Application of PDT-EDOT-2F Nanoparticles

[0065] a) Take 200 μg / mL of PDT-EDOT-2F nanoparticle suspension (prepared in Example 1) and dilute it to 20 μg / mL with deionized water. Take 25 μL of the diluted suspension and drop it onto a 300-mesh ultrathin carbon film (purchased from Sinovac). After drying, observe it using a transmission electron microscope (Hitachi HT7700, operating voltage 200 kV). Figure 3 It can be seen that the particle size of PDT-EDOT-2F nanoparticles is about 13 nm and is relatively evenly dispersed.

[0066] b) A 200 μg / mL suspension of PDT-EDOT-2F nanoparticles was diluted to 20 μg / mL with phosphate buffered saline (PBS, pH 7.4, purchased from Thermo Fisher Scientific). The average hydrated diameter and surface charge of the PDT-EDOT-2F nanoparticles were measured using a dynamic light scattering instrument (Malvern Zetasizer NanoZS ZEN 3600). The test was repeated one week later. Figure 4 It can be seen that the average hydrated diameter of PDT-EDOT-2F nanoparticles is about 213nm, the charge they carry is about -23ev, and the material is relatively stable, with no significant changes in particle size and surface charge within a week.

[0067] c) Absorption and emission spectra characterization and fluorescence quantum yield determination:

[0068] A 20 μg / mL PDT-EDOT-2F nanoparticle suspension obtained by dilution with deionized water was added to a cuvette, and its absorption spectrum was measured using a UV-visible-near-infrared spectrophotometer (Lambda 1050+). Its emission spectrum and fluorescence quantum yield were measured using a steady-state transient fluorescence spectrometer (FLS980).

[0069] In order to calculate the specific fluorescence quantum yield value, the classic NIR-II fluorescent dye IR-1061 was selected as a reference. The IR-1061 dye was diluted with dichloromethane (DCM) into a series of solutions with different concentrations, so that their absorbance values ​​at 808nm were 0.02, 0.04, 0.06, 0.08, and 0.10, respectively. The fluorescence spectra of the above solutions with different concentrations were then tested when excited at 808nm. The corresponding area within the range of 833-1400nm was integrated as the ordinate, and the absorbance value was used as the abscissa. Linear regression analysis was performed to obtain the slope. IR-1061 A similar method was used to obtain the slope corresponding to PDT-EDOT-2F nanoparticles. PDT-EDOT-2F , where PDT-EDOT-2F nanoparticle suspensions of varying concentrations were diluted with chloroform (TCM). To avoid interference from laser excitation, an 850nm longpass filter was used when measuring the emission spectrum. The relative quantum yield of PDT-EDOT-2F nanoparticles was calculated using the following formula:

[0070]

[0071] The quantum yield IR-1061 0.75%, refractive index TCM The refractive index is 1.4711. DCM The absorption spectrum and emission spectrum test results are as follows Figure 5 The fluorescence quantum yield test results are shown in Figure 6 As shown, the fluorescence quantum yield of PDT-EDOT-2F nanoparticles is 5.32%.

[0072] Example 3: Application of PDT-EDOT-2F Nanoparticles

[0073] a) Characterization of near-infrared fluorescence effect in vitro

[0074] 200 μg / mL PDT-EDOT-2F nanoparticle suspension was diluted to 20 μg / mL with water, PBS buffer and FBS solution respectively, and then the suspension was washed with water at a power intensity of about 30 mW / cm 2 The fluorescence images were then recorded using an NIR-II imaging system. Figure 7 shown.

[0075] b) In vivo vascular imaging

[0076] Six-week-old, healthy BALB / c mice (purchased from Beijing Weitong Lihua) were injected via the tail vein with 200 μL of a 20 μg / mL suspension of PDT-EDOT-2F nanoparticles diluted in PBS. The mice were anesthetized and secured on an imaging stage. Whole-body fluorescence angiography was performed using an InGaAs detector equipped with different long-wavelength pass filters (1000, 1100, and 1200 nm). The results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that PDT-EDOT-2F nanoparticles can achieve high-resolution near-infrared zone II vascular imaging in vivo, and compared with the images taken using the 1000nm filter, the near-infrared zone II images using 1100 and 1200nm long-wave pass filters show better contrast and significantly reduced background ( Figure 8 ).

[0077] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Compound of formula (I): In formula (I), R1, R2, R3, and R4 are each independently selected from a hydrogen atom or a C 1-10 alkyl; R5, R6, R7, R8 groups are independently selected from hydrogen atoms, halogen atoms, nitro groups, cyano groups, C 1-6 Halogenated alkyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylcarbonyl, C 1-6 Alkoxycarbonyl, or R6 and R7 together form a 5-14 membered aromatic ring or heteroaromatic ring fused to a benzene ring; wherein the heteroaromatic ring contains 1-3 heteroatoms independently selected from N, O or S; the aromatic ring or heteroaromatic ring is optionally substituted by 1-3 substituents, each of which is independently selected from halogen, nitro, cyano, C 1-6 Halogenated alkyl, C 1-6 Alkylsulfonyl, C 1-6 Alkoxycarbonyl; n is a positive integer from 1 to 4.

2. The compound according to claim 1, characterized in that R1, R2, R3, and R4 are each independently selected from a hydrogen atom or a C 1-8 alkyl; R5, R6, R7, R8 groups are independently selected from hydrogen atoms, halogen atoms, nitro groups, cyano groups, C 1-4 Halogenated alkyl, C 1-4 Alkylsulfonyl, C 1-4 Alkylcarbonyl, C 1-4 Alkoxycarbonyl, or R6 and R7 together form a 5-14 membered aromatic ring or heteroaromatic ring fused to a benzene ring; wherein the heteroaromatic ring contains 1-3 heteroatoms independently selected from N, O or S; the aromatic ring or heteroaromatic ring is optionally substituted by 1-3 substituents, each of which is independently selected from halogen, nitro, cyano, C 1-4 Halogenated alkyl, C 1-4 Alkylsulfonyl, C 1-4 Alkoxycarbonyl; n is a positive integer from 1 to 2.

3. A method for preparing the compound of claim 1 or 2, comprising the steps of reacting a compound of formula II with a compound of formula III under alkaline conditions to obtain a compound of formula (I); In formula II, R1, R2, R3, R4, and n are defined as R1, R2, R3, R4, and n in formula (I); In formula III, the definitions of R5, R6, R7, and R8 are the same as those of R5, R6, R7, and R8 in formula (I).

4. The method according to claim 3, characterized in that The reaction is carried out in an inert atmosphere; The alkaline condition is provided by an organic base, and the organic base can be specifically at least one of pyridine, N,N-dimethylaminopyridine, triethylamine, and N,N-diisopropylethylenediamine; The molar ratio of the compound represented by formula II to the compound represented by formula III is 1:(2-5); The reaction temperature is 85-100°C and the reaction time is 12-24 hours; The reaction is carried out in an organic solvent, and the organic solvent can be specifically chloroform.

5. The method according to claim 3, characterized in that The compound represented by formula II is prepared by a method comprising the following steps: reacting a compound represented by formula IV with a compound represented by formula V in an inert atmosphere in the presence of a palladium catalyst to obtain a compound represented by formula II; In formula IV, R1, R2, R3, and R4 are defined as R1, R2, R3, and R4 in formula (I); In formula V, n is defined as in formula (I).

6. The method according to claim 5, characterized in that The reaction is carried out in an organic solvent, and the organic solvent can specifically be ultra-dry toluene; The inert atmosphere is a nitrogen atmosphere; The palladium catalyst is selected from at least one of palladium acetate, palladium dichloride, tetrakistriphenylphosphine palladium, bis(triphenylphosphine)palladium dichloride, Pd2(dba)3 / PPh3, and Pd(OAc)2 / PPh3; The molar ratio of the compound represented by formula IV, the compound represented by formula V, and the palladium catalyst is 1:(2-5):(0.1-5); The reaction temperature is 80-120° C. and the reaction time is 12-24 hours.

7. A fluorescent material for near-infrared second-zone imaging, comprising the compound represented by formula (I) according to claim 1 or 2, wherein the fluorescent material is a nanoparticle.

8. The fluorescent material according to claim 7, characterized in that The fluorescent material further comprises liposomes, and the liposomes are used to encapsulate the compound represented by formula (I); The mass ratio of the compound represented by formula (I) to the liposome is 1:5-1:

10.

9. Use of the compound of formula (I) according to claim 1 or 2 or the fluorescent material according to claim 7 as and / or in the preparation of a product for near-infrared second-zone imaging.

10. The use according to claim 9, characterized in that In the application, the product is used for in vivo near-infrared zone II vascular imaging.

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